Class II (Ia) gene products play critical roles in a variety of T lymphocyte responses. They are the primary stimulating antigens in allogeneic and syngeneic mixed lymphocyte responses, they "restrict" recognition of foreign antigens by Lyt1+ (L3T4+) T lymphocytes, and they control the ability of animals to respond to T dependent antigens (immune response [Ir] gene function). A combination of immunological and molecular genetic approaches is being used to gain an understanding of the structural basis for this recognition of Ia by T lymphocytes. Towards this goal, genomic or cDNA clones of Aalpha(b,d,k), Abeta(b,d,k,bm12), Ealpha(k,d), Ebeta(d,k) and Ebeta(d,k,) have been isolated, and where necessary, sequenced. AAlpha and ABeta genes have been transfected into B lymphomas or L-cells, and Ia expressing transformants obtained. These have been used to stimulate a variety of T cell hybridomas and clones, establishing the importance of both AAlpha and ABeta polymorphic regions in forming restriction elements. Exon-shuffling between allelic ABeta genes localized both serologic and T cell recognition sites to the highly variable Beta1 domain. Sequence analysis of EMS induced ABeta mutants also localized a critical site of function to a small region of the Beta1 domain near the recently sequenced bm12 mutation. Finally attempts to construct transfectant expressing "hybrid" I-A molecules revealed an unexpected restriction on Alpha:Beta chain assembly which maps to the Beta1 domain. These results have shown the validity of this approach in determining the critical structural features of class II molecules recognized by T lumphocytes, and the molecular basis of Ia chain assembly.